AU2019359620A1 - Efficient composite bacterial agent for degrading petroleum, and preparation method and application thereof - Google Patents
Efficient composite bacterial agent for degrading petroleum, and preparation method and application thereof Download PDFInfo
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- 239000003208 petroleum Substances 0.000 title claims abstract description 93
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 55
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 55
- 239000002131 composite material Substances 0.000 title claims abstract description 30
- 230000000593 degrading effect Effects 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 230000015556 catabolic process Effects 0.000 claims abstract description 68
- 238000006731 degradation reaction Methods 0.000 claims abstract description 68
- 229930195733 hydrocarbon Natural products 0.000 claims abstract description 31
- 150000002430 hydrocarbons Chemical class 0.000 claims abstract description 31
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 27
- 241000216250 Tsukamurella pulmonis Species 0.000 claims abstract description 27
- 241000383873 Sphingopyxis Species 0.000 claims abstract description 26
- 244000005700 microbiome Species 0.000 claims abstract description 10
- 239000002689 soil Substances 0.000 claims description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 21
- 238000011081 inoculation Methods 0.000 claims description 18
- 239000003344 environmental pollutant Substances 0.000 claims description 15
- 231100000719 pollutant Toxicity 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000003209 petroleum derivative Substances 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- 238000009629 microbiological culture Methods 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- 239000003415 peat Substances 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 239000012138 yeast extract Substances 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 239000003337 fertilizer Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 239000011574 phosphorus Substances 0.000 claims description 3
- 239000010802 sludge Substances 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000000411 inducer Substances 0.000 claims description 2
- 238000003971 tillage Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 4
- 230000002195 synergetic effect Effects 0.000 abstract description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 239000007789 gas Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 238000005067 remediation Methods 0.000 description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 229910017053 inorganic salt Inorganic materials 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000007836 KH2PO4 Substances 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 241000589291 Acinetobacter Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000204066 Tsukamurella Species 0.000 description 1
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 238000013329 compounding Methods 0.000 description 1
- 150000001924 cycloalkanes Chemical class 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
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- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000011066 ex-situ storage Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
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- 239000013049 sediment Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
Abstract
Abstract The present invention relates to an efficient composite bacterial agent for degrading petroleum and a preparation method and application thereof. An efficient petroleum degradation composite bacterial agent is provided, comprising the following effective microorganisms: Tsukamurella pulmonis ECO 17 with accession number CGMCC No. 15837; Bacillus paramycoides-3 with accession number CGMCC No. 16238; Sphingopyxis chilensis-13 with accession number CGMCC No. 16241; Achromoobacterpulmonis-10 with accession number CGMCC No. 16240. In the present invention, degradation strains are screened according to the degradation characteristics of the strains to petroleum hydrocarbons. The four strains have different degradation characteristics to petroleum hydrocarbons of different carbon chain lengths. Moreover, the four strains do not competitively inhibit growth after being compounded, and achieve a synergistic promotion effect, so that the degradation efficiency of petroleum hydrocarbons can be improved.
Description
EFFICIENT COMPOSITE BACTERIAL AGENT FOR DEGRADING
PETROLEUM, AND PREPARATION METHOD AND APPLICATION THEREOF
Field of the Invention
The present invention relates to an efficient composite bacterial agent for degrading petroleum and a preparation method and application thereof, and belongs to the technical field of microorganisms.
Background of the Invention
The pollution of oil and polycyclic aromatic hydrocarbons in soil has the characteristics of concealment, latency, irreversibility, long lasting and serious consequence. Bioremediation is an effective remediation method for petroleum-polluted soil. Bioremediation is a controlled or spontaneous process that degrades, consumes, decomposes, converts, metabolizes and/or removes pollutants in the environment by means of biological systems (including microorganisms, plants and enzymes), to achieve the purpose of reducing or ultimately eliminating environmental pollution. In most cases, microbial communities degrading hydrocarbon will grow in petroleum-polluted soil and sediments. If there is sufficient time, the soil of the polluted site will return to its previous state. To achieve such effects, there must be a sufficient number of microorganisms and a large number of key genes for the degradation of petroleum hydrocarbons. However, the remediation time also depends on a series of other factors: (1) pollution degree: high concentrations of petroleum hydrocarbons will inhibit the growth of microorganisms due to their cytotoxicity, particularly petroleum hydrocarbon components with high water-solubility and low hydrophobicity; and, (2) soil condition: to maintain biological activity, bacteria for degrading petroleum hydrocarbons require specific conditions including appropriate pH values, temperature ranges, free oxygen, nutrient elements (nitrogen and phosphorus), oxygen and moisture. If these conditions are not within acceptable limits for microbial growth, microbial degradation of total petroleum hydrocarbons is halted. In this case, the degradation of petroleum hydrocarbons is realized only by chemical and weathering effects, so it may last for a very long time.
The in-situ and the ex-situ bioremediation technologies are common, both of which utilize petroleum hydrocarbons as a carbon source to catalyze and degrade petroleum pollutants through the metabolic process of microorganisms. In China and foreign countries, the research on bioremediation of petroleum-polluted soil has been increasing, and many achievements have been made. The bioremediation of oil-polluted soil has the following remarkable advantages: this method is applied to the remediation of large-area polluted soil, and the cost of this remediation technology is low; the remediation effect on the soil is good, and the residual amount of pollutants in the soil is low; this method is green and environment-friendly, and no secondary pollution will not be caused to soil by this method; and, the soil conditions required by plant growth are not damaged, and the soil quality can be improved.
There are a large number of bacteria degrading petroleum hydrocarbons , including more than 70 genera such as Pseudomonas, Micrococcus, Nocardia and Acinetobacter, and more than 200 species. Since petroleum hydrocarbon components are complex, including straight-chain alkanes, branched-chain alkanes, cycloalkanes, polycyclic aromatic hydrocarbons, nitrogen-containing compounds, sulfur-containing compounds and the like, different degrading bacteria can utilize different petroleum components. Therefore, the degrading effects of compound bacteria and floras on petroleum hydrocarbons are better than that of single bacteria. At present, a common microorganism compounding method is to carry out combination tests on microorganisms with a high petroleum degradation rate and then verify the degradation function of the compounded bacteria after combination so as to screen a compounded combination having better degradation effect than that of single bacteria.
Summary of the Invention
In view of the defects in the prior art, the present invention provides an efficient composite bacterial agent for degrading petroleum and a preparation method and application thereof.
The present invention employs the following technical solutions.
An efficient composite bacterial agent for degrading petroleum is provided, comprising the following effective microorganisms:
Tsukamurella pulmonis ECO-17, deposited on June 1, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 15837;
Bacillus paramycoides-3, deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16238;
Sphingopyxis chilensis-V3, deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16241; and
Achromoobacter pulmonis-10, deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16240.
In accordance with the present invention, preferably, the effective viable count of the Tsukamurella pulmonis ECO-17 is 108 to 1010 CFU/g, the effective viable count of the Bacillus paramycoides-3 is 108 to 1010 CFU/g, the effective viable count of the Sphingopyxis chilensis-\3 is 108 to 1010 CFU/g, and the effective viable count of the Achromoobacterpulmonis-VI is 108 to 1010 CFU/g.
A preparation method of the efficient composite bacterial agent for degrading petroleum is provided, comprising the following steps:
(1) activating and culturing Tsukamurella pulmonis ECO-17, Bacillus paramycoides-3, Sphingopyxis chilensisA3 and Achromoobacter pulmonis-\0, respectively;
(2) performing seed culture on the activated Tsukamurella pulmonis ECO-17, Bacillus paramycoides-3, Sphingopyxis chilensis-\5 and Achromoobacter pulmonis-V) obtained in the step (1), respectively;
(3) mixing, fermenting and culturing the Tsukamurella pulmonis ECO-17 seed solution, Bacillus paramycoides-3 seed solution, Sphingopyxis chilensis-\5 seed solution and AchromoobacterpulmonisAQ bacteria solution obtained in the step (2) to obtain a fermented bacteria solution; and (4) mixing the fermented bacteria solution obtained in the step (3) with peat soil at a mass ratio of 1:(5-20) to obtain a petroleum hydrocarbon degradation bacterial agent.
In accordance with the present invention, preferably, in the step (1), the activation and culture condition is placing upside down and culturing for 1 to 2 days at 28°C to 37°C, and the activation culture medium is an LB solid culture medium containing the following components per liter:
g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, and water added to a constant volume of 1 L, with a natural pH.
In accordance with the present invention, preferably, in the step (2), the seed culture condition is shaking and culturing for 1 to 2 days at 28 °C to 37 °C and at 100 to 200 r/min, and the seed culture medium is an LB liquid culture medium containing the following components per liter:
g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and water added to a constant volume of 1 L, with a natural pH.
In accordance with the present invention, preferably, in the step (3), the mixing and fermentation culture condition is culturing for 1 to 2 days at 28 °C to 37 °C and at a dissolved oxygen concentration of 20% to 70%, and the fermentation culture medium contains the following components per liter:
g of peptone, 5 g of yeast extract, 10 g of sodium chloride, water added to a constant volume of 1 L, and 0.1 %o to l%o of petroleum as an inducer, with a natural pH.
In accordance with the present invention, preferably, in the step (3), the inoculation amount of the Tsukamurella pulmonis ECO-17 is 0.5% to 5% (volume percentage), the inoculation amount of the Bacillus paramycoides-3 is 0.5% to 5% (volume percentage), the inoculation amount of the Sphingopyxis chilensis-\3 is 0.5% to 5% (volume percentage), and the inoculation amount of the Achromoobacter pulmonis-lb is 0.5% to 5% (volume percentage).
In accordance with the present invention, preferably, the step (4) further comprises a step of drying in the air until the water content is 10% to 15% after mixing.
In accordance with the present invention, preferably, in the step (4), the water content of the peat soil is < 5%, and the mass percentage content of organic matters is 40% to 60%.
An application of the efficient composite bacterial agent for degrading petroleum in degrading petroleum pollutants is provided.
In accordance with the present invention, preferably, the application comprises the following steps:
adding the efficient petroleum degradation composite bacterial agent into a petroleum pollutant to be remediated, wherein the addition amount of the efficient petroleum degradation composite bacterial agent is 0.1% to 2% of the mass of the petroleum pollutant; adding a compound fertilizer having a nitrogen-to-phosphorus ratio of 5:1; performing rotary tillage; maintaining the moisture of soil to be 15% to 25%; and, treating for 30 to 180 days.
In accordance with the present invention, further preferably, the petroleum pollutant is soil or sludge polluted by petroleum.
In accordance with the present invention, further preferably, the mass content of petroleum hydrocarbons contained in the petroleum pollutant is 0.5% to 7%.
In accordance with the present invention, further preferably, the addition amount of the compound fertilizer is 0.01% to 0.1% of the mass of the petroleum pollutant.
Beneficial effects
In the present invention, degradation strains are screened according to the degradation characteristics of the strains to petroleum hydrocarbons, and then compounded according to the principle of complementary degradation characteristics. The four strains have different degradation characteristics to petroleum hydrocarbons of different carbon chain lengths, and have remarkable degradation effects on petroleum hydrocarbons of different carbon chain lengths having a carbon number of <14, 14-28, 29-36, >C36 or the like. Moreover, the four strains do not competitively inhibit growth after being compounded, and achieve a synergistic promotion effect, so that the degradation efficiency of petroleum hydrocarbons can be improved.
Brief Description of the Drawings
Fig. 1 is a gas phase graph of petroleum degradation by Tsukamurellapulmonis ECO-17;
hydrocarbons after petroleum
Fig. 2 is a gas phase graph of petroleum degradation by Bacillus paramycoides-3;
hydrocarbons after petroleum
Fig. 3 is a gas phase graph of petroleum degradation by Sphingopyxis chilensisAV, hydrocarbons after petroleum
Fig. 4 is a gas phase graph of petroleum degradation by Achromoobacter pulmonis AC, hydrocarbons after petroleum
Fig. 5 is a gas phase graph of petroleum degradation by an efficient petroleum degradation composite bacterial agent;
hydrocarbons after petroleum
Fig. 6 is a gas phase graph of a petroleum solution; and
Fig. 7 is a gas phase graph of petroleum hydrocarbons after petroleum degradation by a bacterial agent in Comparative Example 1.
Detailed Description of the Embodiments
The technical solutions of the present invention will be further illustrated below by examples, but the protection scope of the present invention is not limited thereto.
Biomaterial sources
Tsukamurella pulmonis ECO-17: deposited on June 1, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 15837.
Bacillus paramycoides-3'. deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16238.
Sphingopyxis chilensis-13'. deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16241.
Achromoobacter pulmonis-10'. deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16240.
Culture mediums
LB solid medium containing the following components per liter:
g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, and dfhO added to a constant volume of 1 L.
LB liquid culture medium containing the following components per liter:
g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and dH2O added to a constant volume of 1 L.
Inorganic salt culture medium containing the following components per liter:
1.5 g of KNO3, 1.5 g of (NH4)2SO4, 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of MgSO4’7H2O, 0.5 g of NaCl, 0.01 g of FeSO4’7H2O, and dH2O added to a constant volume of 1 L.
Petroleum-inorganic salt solid medium containing the following components per liter:
1.5 g of KNO3, 1.5 g of (NH4)2SO4, 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of MgSO4’7H2O, 0.5 g of NaCl, 0.01 g of FeSO4’7H2O, 10% petroleum, 20 g of agar, and dH2O added to a constant volume of 1 L.
Petroleum-inorganic salt liquid culture medium containing the following components per liter:
1.5 g of KNO3, 1.5 g of (NH4)2SO4, 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of MgSO4’7H2O, 0.5 g of NaCl, 0.01 g of FeSO4«7H2O, 10% petroleum, and dH2O added to a constant volume of 1 L.
In the examples, the water content of the peat soil is <5 %, and the mass percentage content of organic matters is 40-60%; and the peat soil is purchased from Shandong Environmental Peat Soil Co., Ltd.
Example 1
The preparation method of the efficient petroleum degradation composite bacterial agent comprised the following steps.
(1) Tsukamurella pulmonis ECO-17, Bacillus paramycoides-3, Sphingopyxis chilensisA3 and Achromoobacter pulmonisAO were activated and cultured, respectively, wherein the activation and culture condition was placed upside down and culturing for 1 day at 32°C.
(2) Seed culture was performed on the activated Tsukamurellapulmonis ECO-17,
Bacillus paramycoides-3, Sphingopyxis chilensis-\5 and Achromoobacter pulmonis-10 obtained in the step (1), respectively, wherein the seed culture condition was shaken and cultured for 1 day at 32°C and at 150 r/min.
(3) The Tsukamurella pulmonis ECO-17 seed solution, Bacillus paramycoides-3 seed solution, Sphingopyxis chilensis-\3 seed solution and Achromoobacter pulmonisAO bacteria solution obtained in the step (2) were mixed, fermented and cultured to obtain a fermented bacteria solution, wherein the mixing and fermentation culture condition was cultured for 1 day at 32 °C and at a dissolved oxygen concentration of 50%.
The fermentation culture medium contained the following components per liter:
g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 0.5 g of petroleum, and dbhO added to a constant volume of 1 L.
The inoculation amount of the Tsukamurella pulmonis ECO-17 was 2% (volume percentage), the inoculation amount of the Bacillus paramycoides-3 was 2% (volume percentage), the inoculation amount of the Sphingopyxis chilensis-\3 was 2% (volume percentage), and the inoculation amount of the Achromoobacter pulmonisAO was 2% (volume percentage).
(4) The fermented bacteria solution obtained in the step (3) was mixed with peat soil at a mass ratio of 1:10, and then naturally dried for 2 h in a shade place until to the water content was 10%, to obtain a petroleum hydrocarbon degradation bacterial agent.
Through detection, in the prepared petroleum hydrocarbon degradation bacterial agent, the effective viable count of the Tsukamurella pulmon was ECO-17, the Bacillus paramycoides-3, the Sphingopyxis chilensis-I3 and the Achromoobacter pulmonisAO was about 2.DIO8 CFU/g, 1.7χ 108 CFU/g, 1.5χ 108 CFU/g and 1.3χ 108 CFU/g, respectively.
Example 2
This example differed from the efficient petroleum degradation composite bacterial agent described in Example 1 in that, in the step (3), the inoculation amount of the Tsukamurella pulmonis ECO-17 seed solution, the Bacillus paramycoides-3 seed solution, the Sphingopyxis chilensis-I3 seed solution and the Achromoobacter pulmonisAQ seed solution was 1%, 1%, 2% and 2% (volume percentage), respectively.
Through detection, in the prepared petroleum hydrocarbon degradation bacterial agent, the effective viable count of the Tsukamurella pulmonis ECO-17, the Bacillus paramycoides-3, the Sphingopyxis chilensisA3 and the Achromoobacter pulmonis-ft was about 1.7^108 CFU/g, 1.4xl08 CFU/g, 2.3><108 CFU/g and 2.1x10s CFU/g, respectively.
Example 3
This example differed from the efficient petroleum degradation composite bacterial agent described in Example 1 in that, in the step (4), the mass ratio of the fermented solution to the peat soil was 1:5.
Through detection, in the prepared petroleum hydrocarbon degradation bacterial agent, the effective viable count of the Tsukamurella pulmonis ECO-17, the Bacillus paramycoides-3, the Sphingopyxis chilensis-\3 and the AchromoobacterpulmonisAQ was about 3.2*108 CFU/g, 2.5><108 CFU/g, 4.3><108 CFU/g and 4.1 χ 10s CFU/g, respectively.
Comparative Example 1
This example differed from the efficient petroleum degradation composite bacterial agent described in Example 1 in that, Achromoobacter pulmonis-\() was replaced with Tsukamurella pulmonis ECO-17. The inoculation amount of each strain in the step (3) in Example 1 was as follows: the inoculation amount of Tsukamurella pulmonis ECO-17 was 4% (volume percentage), the inoculation amount of Bacillus paramycoides-3 was 2% (volume percentage), and the inoculation amount of Sphingopyxis chilensis-\3 was 2% (volume percentage).
The fermented solution was mixed with the peat soil at a mass ratio of 1:10, and then naturally dried in a shade place for 2 h until the water content was 10%, to obtain a petroleum hydrocarbon degradation bacterial agent.
Through detection, in the prepared petroleum hydrocarbon degradation bacterial agent, the effective viable count of the Tsukamurella pulmonis ECO-17, the Bacillus paramycoides-3 and the Achromoobacter pulmonis-ft) was about 3.2><108 CFU/g,
2,ίχ108 CFU/g and 1.9* 108 CFU/g, respectively.
Experimental Example 1
The Tsukamurella pulmonis ECO-17 seed solution, the Bacillus paramycoides-3 seed solution, the Sphingopyxis chilensisA3 seed solution and the Achromoobacter pulmonisAQ bacteria solution prepared in the step (2) in Example 1 were fermented and cultured, respectively, where the culture conditions were the same as the conditions in the step (3) in Example 1. Then, singe bacterial agents were prepared from the fermented solutions of the four bacteria according to the conditions in the step (4) in Example 1, respectively. The viable count in each singe bacterial agent was the same as the total viable count of in the bacterial agent prepared in Example 1. The bacterial agents prepared from the Tsukamurella pulmonis ECO-17 seed solution, the Bacillus paramycoides-3 seed solution, the Sphingopyxis chilensisA3 seed solution and the Achromoobacterpulmonis-lb were labelled as 1#, 2#, 3# and 4#, respectively; the composite bacterial agent described in Example 1 was labelled as 5#; and the bacterial agents 1#, 2#, 3#, 4# and 5# were the same in microbial concentration.
The bacterial agents 1#, 2#, 3#, 4# and 5# were inoculated into petroleum-inorganic salt solid culture mediums at an inoculation amount of 5% by mass percentage, and then cultured for 7d on a shaker at 30°C and at 150 rpm, respectively. A culture medium that was not inoculated with any bacteria was used as a blank control. At the end of culture, the culture solution was added with dichloromethane, the residual petroleum was extracted, the extract solution was added with a sufficient amount of anhydrous sodium sulfate for dehydration, then filtered and rotary-evaporated so that the dichloromethane was completely volatilized; and the system was stood at room temperature, and weighed after the weight was stable. The degradation rate (η%) of petroleum hydrocarbons was calculated by following formula:
η(%) = (ω0-ωχ)/ω0χ100 where ωθ was the residual oil content in the control culture solution, and ωχ was the residual oil content in the tested bacteria culture solution.
The degradation rate of petroleum hydrocarbons in the differently-treated liquid shake flasks were as follows: the degradation rate of 1# was 39.3%, the degradation rate of 2# was 35.6%, the degradation rate of 3# was 33.9%, the degradation rate of
4# was 38%, and the degradation rate of 5# was 49.8%.
The extracts of the bacterial agents 1#, 2#, 3#, 4# and 5# after degradation and the blank control extract were analyzed by gas chromatography, and the results were shown in Figs. 1-6.
Figs. 1-5 are gas phase graphs of the bacterial agents 1# to 5# after petroleum hydrocarbon degradation, respectively; and Fig. 6 is a gas phase graph of the untreated petroleum hydrocarbon solution. Compared with Fig. 6, Figs. 1-4 show better degradation effects on petroleum hydrocarbons, and it can be seen from Figs. 1-4 that different degradation effects are achieved for different carbon chain lengths, and the degradation effects on petroleum hydrocarbons having carbon chain lengths in ranges of <14, 29-36, >C 36 and 14-28 are particularly remarkable. It can be seen from Fig. 5 that the bacterial agent formed by the combination of the four bacteria has a good degradation effect on short-chain and long-chain petroleum hydrocarbons, and the degradation rate of petroleum hydrocarbon is obviously improved.
Experimental Example 2
Sludge-polluted soil remediation tests were performed on the efficient petroleum degradation composite bacterial agent prepared in Example 1 and the bacterial agent in Comparative Example 1. The specific method was described below.
The soil polluted by landing oil was collected, then spread into a thin layer and naturally dried until the water content was 15%, and the oil sludge block was manually crushed. 5 kg of a nitrogen-phosphorus fertilizer and 10 kg of the efficient petroleum degradation composite bacterial agent were added to the polluted soil per ton, the soil was kept loose and piled to form a pile having a height of 50 cm and a width of 100 cm, and the pile was remediated for one month.
The pile was sampled, and the sample was extracted with dichloromethane for three times. The content of petroleum was calculated by the method in Example 4.
Through detection, the petroleum degradation rate of the bacterial agent in Example 1 was 37.4%, and the petroleum degradation rate of the bacterial agent in Comparative
Example 1 was 30.6%.
Fig. 7 is the analysis on the result of remediation of the petroleum-polluted soil by the bacterial agent in Comparative Example 1. By analyzing the test results after 10 degradation, compared with the bacterial agent in Example 1, in the petroleum composition of the soil remediated by the bacterial agent in Comparative Example 1 the residual amount of medium-chain and long-chain petroleum hydrocarbons is obviously increased, indicating that the combination of strains in this bacterial agent does not effectively degrade medium-chain and long-chain petroleum hydrocarbons.
Claims (3)
- Claims1. An efficient petroleum degradation composite bacterial agent, characterized by comprising the following effective microorganisms:Tsukamurella pulmonis ECO-17, deposited on June 1, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 15837;Bacillus paramycoides-3, deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16238;Sphingopyxis chilensis-\3, deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16241; andAchromoobacterpulmonisAO, deposited on August 9, 2018 in the China General Microbiological Culture Collection Center located at Yard No. 3, No. 1 West Beichen Road, Chaoyang District, Beijing, with accession number CGMCC No. 16240.2. The efficient petroleum degradation composite bacterial agent according to claim 1, characterized in that the effective viable count of the Tsukamurella pulmonis ECO-17 is 108 to 1010 CFU/g, the effective viable count of the Bacillus paramycoides-3 is 108 to 1010 CFU/g, the effective viable count of the Sphingopyxis chilensisA3 is 108 to 1010 CFU/g, and the effective viable count of the AchromoobacterpulmonisAO is 108 to 1010 CFU/g.3. A preparation method of an efficient petroleum degradation composite bacterial agent according to claim 1, characterized by comprising the following steps:(1) activating and culturing Tsukamurella pulmonis ECO-17, Bacillus paramycoides-3, Sphingopyxis chilensis-\5 and Achromoobacter pulmonis-\(), respectively;
- (2) performing seed culture on the activated Tsukamurella pulmonis ECO-17, Bacillus paramycoides-3, Sphingopyxis chilensis-\5 and Achromoobacter pulmonis-16 obtained in step (1), respectively;
- (3) mixing, fermenting and culturing the Tsukamurella pulmonis ECO-17 seed solution, Bacillus paramycoides-3 seed solution, Sphingopyxis chilensisA3 seed solution and Achromoobacter pulmonis-\6 bacteria solution obtained in step (2) to obtain a fermented bacteria solution; and (4) mixing the fermented bacteria solution obtained in the step (3) with peat soil at a mass ratio of 1:(5-20) to obtain a petroleum hydrocarbon degradation bacterial agent.4. The preparation method of an efficient petroleum degradation composite bacterial agent according to claim 3, characterized in that, in the step (1), the activation and culture condition is placing upside down and culturing for 1 to 2 days at 28°C to 37°C, and the activation culture medium is an LB solid culture medium containing the following components per liter:10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, and water added to a constant volume of 1 L, with a natural pH.In accordance with the present invention, preferably, in the step (2), the seed culture condition is shaking and culturing for 1 to 2 days at 28°C to 37°C and at 100 to 200 r/min, and the seed culture medium is an LB liquid culture medium containing the following components per liter:10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and water added to a constant volume of 1 L, with a natural pH.5. The preparation method of an efficient petroleum degradation composite bacterial agent according to claim 3, characterized in that, in the step (3), the mixing and fermentation culture condition is culturing for 1 to 2 days at 28°C to 37°C and at a dissolved oxygen concentration of 20% to 70%, and the fermentation culture medium contains the following components per liter:10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, water added to a constant volume of 1 L, and 0.1 %o to l%o of petroleum as an inducer, with a natural pH.6. The preparation method of an efficient petroleum degradation composite bacterial agent according to claim 3, characterized in that, in the step (3), the inoculation amount of the Tsukamurella pulmonis ECO-17 is 0.5% to 5% (volume percentage), the inoculation amount of the Bacillus paramycoides-3 is 0.5% to 5% (volume percentage), the inoculation amount of the Sphingopyxis chilensisA3 is 0.5% to 5% (volume percentage), and the inoculation amount of the Achromoobacter pulmonis-V) is 0.5% to 5% (volume percentage).7. The preparation method of an efficient petroleum degradation composite bacterial agent according to claim 3, characterized in that the step (4) further comprises a step of drying in the air until the water content is 10% to 15% after mixing.8. The preparation method of an efficient petroleum degradation composite bacterial agent according to claim 3, characterized in that, in the step (4), the water content of the peat soil is < 5%, and the mass percentage content of organic matters is 40% to 60%.9. An application of an efficient petroleum degradation composite bacterial agent according to claim 1 in degrading petroleum pollutants.5 10. The application according to claim 9, characterized by comprising the following steps:adding the efficient petroleum degradation composite bacterial agent into a petroleum pollutant to be remediated, wherein the addition amount of the efficient petroleum degradation composite bacterial agent is 0.1% to 2% of the mass of the 10 petroleum pollutant; adding a compound fertilizer having a nitrogen-to-phosphorus ratio of 5:1; performing rotary tillage; maintaining the moisture of soil to be 15% to 25%; and, treating for 30 to 180 days;further preferably, the petroleum pollutant is soil or sludge polluted by petroleum;15 further preferably, the mass content of petroleum hydrocarbons contained in the petroleum pollutant is 0.5% to 7%; and further preferably, the addition amount of the compound fertilizer is 0.01% to 0.1% of the mass of the petroleum pollutant.
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| CN110317741B (en) * | 2019-03-22 | 2021-09-28 | 厦门大学 | Chromium-resistant petroleum hydrocarbon degrading bacterium Thp3-45A and application thereof |
| CN110592069A (en) * | 2019-09-26 | 2019-12-20 | 中海石油环保服务（天津）有限公司 | Preparation method of petroleum hydrocarbon degrading bacteria immobilized microbial inoculum and immobilized microbial inoculum |
| CN111705000A (en) * | 2020-07-23 | 2020-09-25 | 桂林理工大学 | Method for rapidly screening extractable petroleum hydrocarbon degrading bacteria and application thereof |
| CN111793592A (en) * | 2020-07-23 | 2020-10-20 | 桂林理工大学 | Method for comparing petroleum hydrocarbon degradation capability of different strains and application thereof |
| CN112642857A (en) * | 2020-11-18 | 2021-04-13 | 山东高科联合环保科学研究院有限公司 | Marine microorganism remediation method for petroleum-polluted soil |
| CN113403235B (en) * | 2021-02-01 | 2022-05-03 | 山东大学 | High-temperature-resistant petroleum hydrocarbon degrading bacteria, degrading bacteria composition, degrading bacteria microbial inoculum and application thereof |
| CN113293157A (en) * | 2021-06-18 | 2021-08-24 | 华东理工大学 | Ferroferric oxide modified charcoal immobilized polycyclic aromatic hydrocarbon degrading microbial inoculum and preparation and application thereof |
| CN114480157B (en) * | 2021-07-08 | 2023-08-18 | 西安文理学院 | Acidogenic bacteria NQ-P6, application thereof and culture and identification method of acidogenic bacteria NQ-P6 |
| CN113526651B (en) * | 2021-07-14 | 2022-04-01 | 南开大学 | Working electrode for degrading petroleum hydrocarbon, device for degrading petroleum hydrocarbon and application |
| CN114644994A (en) * | 2021-09-17 | 2022-06-21 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Petroleum hydrocarbon degradation compound microbial agent and preparation method and application thereof |
| CN113817638A (en) * | 2021-09-23 | 2021-12-21 | 大连大学 | Complex microbial inoculant for degrading petroleum pollutants and ecological construction method thereof |
| CN113801820A (en) * | 2021-09-26 | 2021-12-17 | 广东中微环保生物科技有限公司 | Method for screening petroleum degrading bacteria, immobilization method and application of immobilized bacteria agent |
| CN114350537A (en) * | 2021-11-22 | 2022-04-15 | 辽宁中博生态环境技术有限公司 | Electrically activated halohydrocarbon degrading bacterium and application thereof |
| CN115072880B (en) * | 2021-11-29 | 2024-01-26 | 江苏宜裕环保科技有限公司 | Biological flocculant and application thereof |
| CN117566998B (en) * | 2024-01-15 | 2024-04-05 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Polyether demulsifier for high-water-content oil-containing sludge, preparation method and application |
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